INTRODUCTION
Point mutations are the most common events in human genetic diseases and
nearly 50% of disease-associated mutations are C>T and
G>A substitutions (Gaudelli et
al., 2017). Animal modeling of human genetic diseases are valuable in
study of pathogenic mechanism and testing of drug efficacy. CRISPR Cas9
system is an adaptive immune system in bacteria that protects its genome
from invading viruses (Rath, Amlinger,
Rath, & Lundgren, 2015; Sontheimer &
Barrangou, 2015). CRISPR Cas9 system has been successfully applied to
genetic engineering in other cells and organisms. It is as well utilized
to precisely correct or introduce point mutations via homology-directed
repair (HDR). It requires DNA double-strand breaks (DSBs) at the target
and a DNA template with homologous arms
(Wu et al., 2018;
Y. Yang et al., 2016;
L. Yang et al., 2013; Yin et al., 2014).
However, cells respond to DSBs more often with nonhomologous end joining
(NHEJ) that may introduce insertions or deletions (indels) and lead to
disruption of corresponding genes (Davis &
Chen, 2013; Komor et al., 2017).
CRISPR/Cas9-based cytidine base editors (CBEs) have recently been
developed to generate precise base changes with high efficiency
(Gaudelli et al., 2017;
Komor, Kim, Packer, Zuris, & Liu, 2016;
Nishida et al., 2016;
Ma et al., 2016). CBEs system consists of
a CRISPR-Cas9-derived DNA-binding module and a cytidine deaminase, and
is able to introduce nucleotide substitutions of C>T
(Xie et al., 2019;
K. Kim et al., 2017;
D. Kim et al., 2017) and G>A
(Zhen Liu et al., 2018) at targeted loci
without need of DSBs. It has been demonstrated successfully in various
organisms (D. Kim et al., 2017).
Base editing systems have gone through various stage of improvement to
broaden their applicability and utility in editing of single nucleotide
and have been widely applied to cell lines, various animals and plants.
The fourth generation of base editor 4 (BE4) has a cytidine deaminase
(rAPOBEC1) with two copies of uracil glycosylase inhibitor (UGI) that
are directly fused to C terminus of Cas9n, a Cas9 mutant with a D10A
amino acid substitution, through a 32 amino acid linker (Fig. 1A). BE4
enables direct conversion of cytidine (C) to uridine (U) in chosen bases
of DNA sequence (Komor et al., 2017).
However, feasibility and efficacy of this system has not been assessed
in mice. In the current study, we have confirmed that BE4 system is able
to perform a multiplexed base editing with high precision and efficiency
in mice. BE4 system shows great potentials in modeling human genetic
diseases.